Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
One of the major difficulties facing the horticultural and agricultural industries is the control of infestation and resulting damage by pathogens such as fungal pathogens. Plant pathogens account for millions of tonnes of lost production on an annual basis. Although fungicides and other anti-pathogenic chemical agents have been successfully employed, there is a range of environmental and regulatory concerns with the continued use of chemical agents to control plant pests. Furthermore, the increasing use of chemical pesticides is providing selective pressure for the emergence of resistance in populations of pests. There is clearly a need to develop alternative mechanisms of inducing resistance in plants to pathogens such as fungi, insects, microorganisms, nematodes, arachnids, protozoa and viruses.
The plant innate immune system comprises both constitutive or pre-formed and inducible components. Pre-formed immunity includes various physical barriers such as wax layers on leaves and rigid cell walls as well as expression of various antimicrobial proteins (Nurnberger et al. (2004) Immunol Rev 198:249-266). The inducible response can include fortification of the cell wall (Showalter (1993) Plant Cell 5(1):9-23) as well as up-regulation of secondary metabolites (Metlen et al. (2009) Plant Cell Environ 32(6):641-653) and antimicrobial proteins (Berrocal-Lobo et al. (2002) Plant Physiol 128(3):951-961; Li and Asiegbu (2004) J Plant Res 117(2):155-162) which occurs in response to various biotic and abiotic stimuli. These responses can occur locally at the site of infection or in distant, uninfected parts of the plant to produce a systemic response. Inducible immunity can also occur via a gene-for-gene response whereby pathogen-associated molecular patterns (PAMPS) are recognized by specific pattern recognition receptors (PRRs) resulting in a hypersensitive response that prevents further spread of the pathogen (see Jones and Dangl (2006) Nature 444(7117):323-329).
Small, disulfide-rich proteins play a large role in both the constitutive and inducible aspects of plant immunity. They can be categorized into families based on their cysteine arrangements and include the thionins, snakins, thaumatin-like proteins, havein- and knottin-type proteins, lipid transfer proteins and cyclotides as well as defensins.
Plant defensins are small (45-54 amino acids), basic proteins with four to five disulfide bonds (Janssen et al. (2003) Biochemistry 42(27):8214-8222). They share a common disulfide bonding pattern and a common structural fold, in which a triple-stranded, antiparallel β-sheet is tethered to an α-helix by three disulfide bonds, forming a cysteine-stabilized αβ motif (CSαβ [see FIG. 1]). A fourth disulfide bond also joins the N- and C-termini leading to an extremely stable structure. A variety of functions have been attributed to defensins, including anti-bacterial activity, protein synthesis inhibition and α-amylase and protease inhibition (Colilla et al. (1990) FEBS Lett 270(1-2):191-194; Bloch and Richardson (1991) FEBS Lett 279(1):101-104). Plant defensins have been expressed in transgenic plants, resulting in increased resistance to target pathogens. For example, potatoes expressing the alfalfa defensin (MsDef1, previously known as alfAFP) showed significant resistance against the fungal pathogen Verticillium dahliae compared to non-transformed controls (Gao et al. (2000) Nat Biotechnol 18(121:1307-1310). Expression of a Dahlia defensin (DmAMP1) in rice was sufficient to provide protection against two major rice pathogens, Magnaporthe oryzae and Rhizoctonia solani (Jha et al. (2009) Transgenic Res 18(11:59-69).
Despite their conserved structure, plant defensins share very little sequence identity, with only the eight cysteine residues completely conserved. The cysteine residues are commonly referred to as “invariant cysteine residues”, as their presence and location are conserved amongst defensins. Based on sequence similarity, plant defensins can be categorized into different groups (see FIG. 2). Within each group, sequence homology is relatively high whereas inter-group amino acid similarity is low. The anti-fungal defensins from distinct groups appear to act via different mechanisms.
Plant defensins can be divided into two major classes. Class I defensins consist of an endoplasmic reticulum (ER) signal sequence followed by a mature defensin domain. Class II defensins are produced as larger precursors with C-terminal pro-domains or pro-peptides (CTPPs) of about 33 amino acids. Most of the Class II defensins identified to date have been found in solanaceous plant species. An alignment of Class II solanaceous defensins is provided in FIG. 3. NsD1 and NsD2 referred to in FIG. 3 represent novel defensins identified in accordance with the present disclosure. Their inclusion in FIG. 3 is not to imply they form part of the prior art.
Class II solanaceous defensins display anti-fungal activity and are expressed in floral tissues. They include NaD1, which is expressed in high concentrations in the flowers of ornamental tobacco Nicotiana alata (Lay et al. (2003) Plant Physiol 131(3):1283-1293). NaD1 is the only Class II solanaceous defensin for which the mechanism of anti-fungal activity has been investigated. The activity of this peptide involves binding to the cell wall, permeabilization of the plasma membrane and entry of the peptide into the cytoplasm of the hyphae (van der Weerden et al. (2008) J Biol Chem 283(21):14445-14452). Unlike many other defensins, NaD1 appears to be specific for filamentous fungi and has no effect on the growth of yeast, bacteria or mammalian cells.
Expression of NaD1 in cotton enhances the resistance to the fungal pathogens Fusarium oxysporum f.sp. vasinfectum and Verticillium dahliae. Under field conditions, plants expressing NaD1 were twice as likely to survive as untransformed control plants and the lint yield per hectare was doubled. Despite this, there was still a significant level of disease in the NaD1-expressing plants.
The structure of defensins consists of seven ‘loops’, defined as the regions between cysteine residues. Loop 1 encompasses the first β-strand (1A) as well as most of the flexible region that connects this β-strand to the α-helix (1B) between the first two invariant cysteine residues. FIG. 5 shows the loop structure of NaD1 including the conserved cysteine residues. Loops 2, 3 and the beginning of 4 (4A) make up the α-helix, while the remaining loops (4B-7) make up β-strands 2 and 3 and the flexible region that connects them (β-hairpin region). This hairpin region of plant defensins forms a γ-core motif that is found in many anti-microbial peptides of diverse classes (Yount and Yeaman (2005) Protein Pept Lett 12(1):49-67).
This β-hairpin region appears to be essential for the biological activity of plant defensins. Mutations in this region of the radish defensin RsAFP2 (See FIG. 2) generally had a negative impact on its anti-fungal activity. In fact, eight out of the twelve residues identified as essential for anti-fungal activity are located in this region (De Samblanx et al. (1997) J Biol Chem 272(2):1171-1179). Furthermore, a chemically synthesized peptide corresponding to this region of the molecule also has anti-fungal activity on its own (Schaaper et al. (2001) J. Pept. Res. 57(5):409-418). In a separate study, the six residues located in loop 5 of VrD2, a defensin from Vigna radiata, were shown to be essential for its α-amylase inhibitory activity (Lin et al. (2007) Proteins 68(2):530-540). A third study investigated the activity of chimeric proteins containing regions from a defensin with anti-fungal activity (MsDef1) and one without (MtDef2). Chimeric defensins that contained the β2-β3 hairpin region of MsDef1 had almost the same activity as the full MsDef1 protein and chimeric defensins that contained this region from MtDef2 had no activity (Spelbrink et al. (2004) Plant Physiol 135(4):2055-2067).
A flexible loop connecting the first β-strand and the α-helix located adjacent and N-terminal of the second invariant cysteine residue (Loop 1B) has been reported to play a minor role in the anti-fungal activity in some defensins when associated as a patch with residues from Loop5. A mutagenesis study of RsAFP2 identified two amino acids important for activity that were located in this region (De Samblanx et al, 1997 supra). However, when this region of the anti-fungal defensin MsDef1 was replaced with the corresponding region from the non-anti-fungal defensin, there was only a modest change in anti-fungal activity (Spelbrink et al, 2004 supra).
Class II solanaceous defensins have variable degrees of activity against fungi. Some Class I defensins exhibit very low anti-fungal activity. Attempts to modify the defensins to improve and broaden their anti-pathogen activity have hitherto been largely unsuccessful. Development of resistance to some defensins is also a potential problem. There is a need to develop protocols to manipulate the level and spectrum of anti-pathogen activity of defensins. The creation of a range of novel defensins with antipathogen activity also facilitates combating the development of resistance.